Vitamin D and vitamin K1 as novel inhibitors of biofilm in Gram-negative bacteria

Background The persistent surge in antimicrobial resistance represents a global disaster. The initial attachment and maturation of microbial biofilms are intimately related to antimicrobial resistance, which in turn exacerbates the challenge of eradicating bacterial infections. Consequently, there is a pressing need for novel therapies to be employed either independently or as adjuvants to diminish bacterial virulence and pathogenicity. In this context, we propose a novel approach focusing on vitamin D and vitamin K1 as potential antibiofilm agents that target Gram-negative bacteria which are hazardous to human health. Results Out of 130 Gram-negative bacterial isolates, 117 were confirmed to be A. baumannii (21 isolates, 17.9%), K. pneumoniae (40 isolates, 34.2%) and P. aeruginosa (56 isolates, 47.9%). The majority of the isolates were obtained from blood and wound specimens (27.4% each). Most of the isolates exhibited high resistance rates to β-lactams (60.7–100%), ciprofloxacin (62.5–100%), amikacin (53.6–76.2%) and gentamicin (65-71.4%). Approximately 93.2% of the isolates were biofilm producers, with 6.8% categorized as weak, 42.7% as moderate, and 50.4% as strong biofilm producers. The minimum inhibitory concentrations (MICs) of vitamin D and vitamin K1 were 625–1250 µg mL-1 and 2500–5000 µg mL-1, respectively, against A. baumannii (A5, A20 and A21), K. pneumoniae (K25, K27 and K28), and P. aeruginosa (P8, P16, P24 and P27) clinical isolates and standard strains A. baumannii (ATCC 19606 and ATCC 17978), K. pneumoniae (ATCC 51503) and P. aeruginosa PAO1 and PAO14. Both vitamins significantly decreased bacterial attachment and significantly eradicated mature biofilms developed by the selected standard and clinical Gram-negative isolates. The anti-biofilm effects of both supplements were confirmed by a notable decrease in the relative expression of the biofilm-encoding genes cusD, bssS and pelA in A. baumannii A5, K. pneumoniae K28 and P. aeruginosa P16, respectively. Conclusion This study highlights the anti-biofilm activity of vitamins D and K1 against the tested Gram-negative strains, which emphasizes the potential of these vitamins for use as adjuvant therapies to increase the efficacy of treatment for infections caused by multidrug-resistant (MDR) strains and biofilm-forming phenotypes. However, further validation through in vivo studies is needed to confirm these promising results. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-024-03293-6.

Vitamin D and vitamin K1 as novel inhibitors of biofilm in Gram-negative bacteria Background Antimicrobial resistance (AMR) represents a massive threat to public health worldwide.The burden of AMR is tremendous, impacting not only the economic status of patients but also their health and lives [1].Both counteracting drug availability and patient noncompliance with the use of antimicrobial agents assist in the development of resistance and increasing morbidity and mortality in developing countries [2,3].As AMR continues to evolve, last resort antimicrobial agents are now unable to treat many resistant strains reducing the available treatment options [4].
Biofilms consist of communities of microbial sessile (sedentary) cells that attach to abiotic or biotic solid surfaces or air-liquid interfaces.Bacterial biofilms are composed of a matrix of extracellular polymeric substances (EPSs) in which they multiply.This matrix comprises intercellular polysaccharides, proteins and extracellularly released DNA [5,6].Mature biofilm-embedded cells are a thousand times more resistant to antibiotics than their planktonic counterparts are.This is due to the limited penetration of antimicrobial agents through the biofilm matrix and the ability of biofilm cells to upregulate efflux pumps and other resistance genes [7].Hence, within the human body, this complex microbial biofilm provides an environment preventing host immune defences and inactivating a massive number of conventional antimicrobial agents, leading to treatment strategy failure and prolonged hospitalization in clinical settings [8].
Acinetobacter baumannii (A.baumannii), Klebsiella pneumoniae (K.pneumoniae) and Pseudomonas aeruginosa (P.aeruginosa) are the most common Gram-negative bacteria and have high biofilm abilities and multiple resistance to different antibiotic classes according to the World Health Organization (WHO) ranking of multidrug resistant (MDR) pathogens [9].Additionally, A. baumannii, K. pneumoniae and P. aeruginosa are members of a bacterial pathogenic group called ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp), which have the ability to escape the microbicidal action of antimicrobial agents, express an arsenal of virulence factors, invade the immune system and cause life-threatening infections, particularly in immunocompromised individuals [10,11].Severe health complications include bacteraemia, ventilator-associated pneumonia, urinary tract infection, skin and soft tissue infections, neonatal sepsis, and complicated intra-abdominal and burn infections [12].
Approximately 65% of bacterial infections and 80% of chronic nosocomial diseases are attributed to biofilm impenetrability and subsequent drug resistance especially on biological tissue surfaces and medical devices such as respiratory supports and catheters [13].Therefore, there is an urgent need to discover new approaches to combat A. baumannii, K. pneumoniae and P. aeruginosa biofilmrelated infections since the traditional and even newer antibacterial agents are insufficient for eradicating colonized bacterial infections.
The application of vitamins in treating MDR biofilms is receiving increased amount of attention.For instance, vitamin K3 (menadione)-coated sutures have been shown to be effective as powerful biofilm inhibitors against staphylococcal-associated surgical site infections [14].In addition, ascorbic acid (vitamin C) has shown promising activity as an antibacterial and antibiofilm agent against carbapenem-resistant hypervirulent K. pneumoniae, P. aeruginosa, Proteus mirabilis and uropathogenic E. coli [15][16][17][18].A significant reduction in biofilm density caused by a wide panel of human pathogens was also verified through the use of vitamin E [19].Vitamin D is a fundamental supplement obtained either from the diet or by skin synthesis upon exposure to sunlight.It aids in the absorption of many minerals including magnesium, iron, zinc and phosphate which are responsible for the production of rigid protective tissues such as dental enamel.Vitamin D is linked to the disruption of dental plaque, a naturally occurring biofilm [20].Furthermore, many reports have shown a significant correlation between lower serum vitamin D levels and chronic dental caries in children [21][22][23].Vitamin D has also aided in fighting bacterial infections caused by S. aureus [24], P. aeruginosa [25] and Helicobacter pylori [26] either through triggering macrophage-mediated clearance or by reversing the efflux system, thereby restoring antibiotic activity.However, no studies have been performed to assess the effect of vitamin D (cholecalciferol) or vitamin K1 (phytomenadione/phylloquinone) on biofilm formation.Therefore, the present study aims to evaluate the activity of WHO-approved vitamin D and vitamin K1 as potential novel therapies to combat biofilm formation in Gram-negative organisms.The impact of both vitamins on bacterial attachment and mature biofilms was examined.Additionally, their effects on the expression levels of the biofilm regulatory genes in the tested isolates were assessed.

Bacterial strains
A total of 130 Gram-negative isolates of Acinetobacter, Klebsiella and Pseudomonas were clinically isolated from different sources such as blood, wounds, urine, bronchoalveolar lavage (BAL), sputum, burns and ear swabs.All specimens were obtained from the Al-Qasr Al-Aini Hospital, Cairo Hospital and Mansoura University Hospital and handled with the approval of the Research Ethics Committee, Faculty of Pharmacy, Mansoura University.The bacterial standard strains A. baumannii (ATCC 19606 and ATCC17978), K. pneumoniae (ATCC 51503), and P. aeruginosa (PAO1 and PAO14) were also included in this study.Based on microbiological laboratory standards, all the isolates were identified colony morphology on MacConkey's agar media (Oxoid, UK).Reddish-pink colonies of Acinetobacter were selectively picked from chromogenic CHROM™ agar (Pioneer, France, Paris).Green P. areugionsa colonies were picked from cetrimide agar.The IMVic (indole, methyl red, Voges-Proskauer, and citrate utilization) test was also conducted for further confirmation of Klebsiella and Pseudomonas isolates [27].The purified isolates were preserved in a 25% v/v glycerol stock at -80 °C.
Molecular detection of A. baumannii clinical isolates.
Multiplex polymerase chain reaction (multiplex PCR) was carried out to detect recA gene (characteristic of the Acinetobacter genus) and 16-23 S rRNA gene intergenic spacer (ITS) region which is specific for A. baumannii spp [28].In this regard, genomic DNA was first extracted by suspending fresh bacterial colonies in sterile nucleasefree water, after which the PCR tubes were boiled for 10 min at 95 ℃ [29,30].The PCR included the following cycling procedure: 12.5 µl of 2X Dream Taq™ Green PCR Master Mix (Thermo Scientific, USA), 0.5 µl of each forward and reverse primer (Table 1), and 1 µl of extracted DNA.The volume was adjusted with nuclease-free water to a final volume of 25 µl.The addition of nuclease-free water instead of bacterial lysate to the other PCR mixture served as a negative control.The cycling procedure consisted of initial denaturation at 94 ℃ for 3 min; 35 cycles of denaturation at 94 ℃ for 30 s and annealing for 30 s at 60 ℃ and termination of the reaction by a final extension step at 72 ℃ for 10 min.After staining with ethidium bromide, two bands at the correct predicted size for recA (425 bp) and 16-23 S rRNA ITS (208 bp) were visualized on an agarose gel (1.5% w/v) which indicated positive A. baumannii isolates.

Detection of biofilm formation
Biofilm formation of all clinical isolates was assessed using a flat-bottom 96-well polystyrene microtiter plate assay [33,34].A. baumannii, K. pneumoniae and P. aeruginosa isolates were propagated overnight in tryptic soy broth (TSB) at 37 °C with shaking at 200 rpm.The wells were filled in sextuplicate with 200 µl of each bacterial culture diluted to the 0.5 McFarland standard.Negative control wells seeded with 200 µl of TSB only were also included.The plates were incubated overnight at 37 °C, after which the bacterial cultures were gently aspirated to  , where ODc (cut-off OD) was defined as 3 standard deviations above the mean OD of the negative control [34,35].

Evaluation of the minimum inhibitory concentrations (MICs) of vitamin D and Vitamin K1
The minimum inhibitory concentrations (MICs) of vitamin D and vitamin K1 were determined using 96-well flat bottom microtiter plates according to the CLSI broth microdilution protocol [32].The MICs of both vitamins were evaluated against selected Gram-negative isolates known for strong biofilm production, including A. baumannii standard strains (ATCC 19606 and ATCC 17978) and clinical isolates (A5, A20 and A21), K. pneumoniae standard strains (ATCC 51503) and clinical isolates (K25, K27 and K28), in addition to P. aeruginosa PAO1 and PAO14 standard strains and P8, P16, P24 and P27 clinical isolates.The Muller-Hinton broth (MHB) was used to propagate the tested isolates overnight at 37 °C, after which the cultures were diluted to a final concentration of 1 × 10 6 CFU/ml.One hundred microliters of injectable vitamin D (Memphis Company) (1.25 mg/ml) and vitamin K1 (Amoun Pharmaceutical Co. S.A.E.) (10 mg/ml) were prepared as twofold serial dilutions with MHB in 96-well microtiter plates.Then, 10 µl of the diluted bacterial suspension was added to each well to reach a final concentration of 10 5 CFU ml − 1 and the plates were incubated at 37 °C under aerobic conditions for 24 h.Nontreated bacteria and MHB broth medium were used as positive and negative controls, respectively.The MICs were calculated as the lowest concentration of vitamin D or K1 that inhibited bacterial growth and sub-MICs (1/2 MIC and 1/4 MIC) were calculated for further experiments.

Effect on cell growth kinetics
The effect of vitamin D and vitamin K1 on the growth of three bacterial isolates, A. baumannii A5, K. pneumoniae K28 and P. aeruginosa P16, was estimated by direct optical density measurements.A single pure colony of each isolate was inoculated in Luria-Bertani (LB) broth and shaken (150 rpm) at 37 °C.The overnight culture was inoculated (1 × 10 6 CFU/ml) into two preparations: untreated and treated with a 1/2 MIC of each tested vitamin.All the preparations were incubated at 37 °C with agitation at 120-150 rpm and the sample (200 µl) was aspirated at 2 h intervals for 24 h, after the bacterial growth was at OD600 nm via ELISA spectrophotometer (BioTek, USA).

Effect of vitamins on cell viability
The viable bacterial cell number was evaluated by counting the colony forming units (CFUs) before and after treatment with 1/2 MICs of vitamins D and K1.At each time interval from the previous procedure, tenfold serial dilution in LB broth was performed for each suspension.Agar plates were prepared and divided into four quadrants.Five microliter from each dilution in the series was applied in each corner per quadrant.The plates were kept at room temperature until complete inoculum drying and incubated in an inverted position at 37 °C for 24 h.Viable cells were calculated in terms of CFUs/ml according to the following formula: CFU/ml= (average number of colonies x dilution factor)/ the inoculum volume on agar plate (ml).The data are expressed as log CFU/ml.

Antibiofilm activities of vitamin D and vitamin K1 Inhibition of initial bacterial attachment
The effect of vitamin D and vitamin K1 on bacterial adherence and initial biofilm configuration was evaluated using the crystal violet (CV) technique [36,37].
Both standard and strong biofilm-producing clinical isolates, as previously selected, were propagated in TSB at 37 °C.The next day, each bacterial culture was diluted to 0.5 McFarland turbidity and treated with each vitamin at sub-MICs (1/2 MIC and 1/4 MIC).The wells of the microtiter plate were seeded with untreated and treated cultures (200 µl each), including negative control wells containing TSB only.The plates were incubated overnight at 37 °C.The biofilm-forming ability of the tested isolates was determined with a CV assay, as described subsequently.

Mature biofilm inhibition assay
The effects of vitamins D and K1 were estimated against preexisting biofilms of standard strains and previously identified clinical isolates, which were powerful biofilm producers.On flatted-bottom 96-well polystyrene plates, 200 µl of each tested isolate (final cell concentration = 1.5 × 10 8 CFU mL − 1 ) was added to each well.Negative control wells containing only TSB were also included.
After overnight incubation at 37 °C to allow biofilm maturation, unattached planktonic cells were aspirated, and the wells were rinsed twice with saline solution.Two hundred microliters of sterile TSB was added to the negative control wells, and another TSB mixture supplemented with 0.5×, 1×, and 2× the MIC of both vitamin D and K1 was added to the conformable wells.The plates were then reincubated at 37 °C for 24 h, and the effect of variable concentrations of both vitamins on the mature biofilm was detected using CV assays, as described below [36].

Quantification of biofilms by crystal violet (CV) assay
Following static overnight incubation of the plates with and without varying concentrations of vitamins, the planktonic cells were aspirated, leaving the adhered biofilm, which was washed twice and fixed with methanol (250 µl) for 15 min.After removal of excess methanol, the plates were allowed to dry before biofilm staining with CV (1% w/v).Excess dye was gently rinsed under tap water, and the adhered biofilms were quantified through solubilization in 250 µl of glacial acetic acid (33% v/v).The absorbance was further measured at 570 nm using a microtiter plate reader, and the percentage of biofilm formation in vitamin D-and K1-treated cultures was calculated compared to that in unchallenged cultures [38,39].

Genotypic analysis
Three housekeeping genes, rpoB, ropD and ropD, in addition to three other biofilm-related genes, cusD, bssS and pelA, specific for A. baumannii (A5), K. pneumoniae (K28) and P. aeruginosa (P16), respectively were investigated by PCR.As previously mentioned, genomic DNA was extracted, and simplex PCR was performed in a 25 µl reaction mixture using the primers and annealing temperatures detailed in Table 1.The PCR program included an initial denaturing cycle at 95 °C for 5 min, followed by (denaturation at 95 °C for 30 s, annealing for 30 s and extension at 72 °C for 1 min) for 35 cycles and a final extension cycle at 72 °C for 5 min.Agarose gel electrophoresis (1.5% w/v) was used to visualize the successful amplification of the PCR product according to the size of the amplicon, as demonstrated in Table 1, and the gel images were captured using a gel documentation system (Model Gel Documentation 1.4, 1189, AccuLab, New York, USA).

Real-time PCR assay
The effect of vitamin D and vitamin K1 on the expression of the biofilm genes cusD, bssS and pelA was assessed through real-time polymerase chain reaction (RT-PCR).
The clinical isolates A5, K28 and P16 were cultivated under the same conditions in the presence and absence of (1/2 MIC) both vitamins.Cultures were subjected to shaking at 150 rpm for 5-6 h until the logarithmic phase of growth was reached.Next, the cultures were centrifuged at 4 ℃ for 20 min at 8000 rpm to collect the bacterial cells, and total RNA was extracted using TRIzol reagent (Oxoid, Basingstoke, Hants, UK) according to the manufacturer's instructions.cDNA was synthesized using a Thermo Scientific RevertAid First Strand cDNA Synthesis Kit.In addition, the concentration and purity of the extract were estimated using a Nanodrop (BioDrop, UK).Any contaminating DNA was removed using DNase enzyme (Enzymonics, Korea) according to the manufacturer's instructions, followed by precipitation of pure mRNA with an equal volume of isopropanol and double washing with ethanol (75% v/v).After complete removal of the ethanol, the mRNA pellets were resuspended in 20 µl of RNase/DNase-free water (Sigma Aldrich, UK).RT-PCR analysis was performed using HERA Plus SYBR Green on a Rotor-Gene Q thermocycler (Qiagen, Valencia, CA, USA) utilizing the biofilm-specific primers listed in Table 1.The relative expression levels of cusD, bssS and pelA genes were normalized to the expression levels of rpoB, ropD, and ropD housekeeping genes in A. baumannii K. pneumoniae and P. aeruginosa, respectively, in accordance with the 2 −∆∆CT analysis method [40].Comparison of gene expression, in terms of fold change, between untreated and vitamin-treated cultures was also performed.

Statistical analysis
An Excel sheet was used to calculate the average and standard deviation of each experiment, which was conducted in triplicate.GraphPad Prism software (version 5.00) was used for all the statistical analyses.The chisquare test was utilized for intergroup comparisons in addition to comparisons between treated and untreated control isolates cultivated under the same circumstances, assuming that P < 0.05 reflected significant data.

Isolation and identification of Gram-negative isolates
Out of the 130 Gram-negative bacterial collections, 117 isolates were phenotypically and biochemically identified and confirmed to be A. baumannii (21 isolates, 17.9%), K. pneumoniae (40 isolates, 34.2%) and P. aeruginosa (56 isolates, 47.9%).For further validation of the A. baumannii isolates, multiplex PCR of recA and 16-23 S rRNA genes showed that all the phenotypically identified isolates had the desired genes at 425 and 208 bp, respectively (Additional file 1. Supplementary Fig. 1).
Based on CLSI interpretive criteria, isolates that were resistant to three or more antibiotic classes were considered MDR.Notably, MDR strains accounted for 82 (70.1%) of the total Gram-negative isolates.Significantly, the highest proportion of MDR bacteria was A. baumannii (81%, P = 0.0001), which was mostly observed among the urine samples, while the lowest was P. aeruginosa (62.5%, P = 0.0463) among the wound samples.The MDR rate among K. pneumoniae was 75% (P = 0.0029), and most of the isolates were from blood sources (Fig. 2d).

Minimum inhibitory concentrations of the tested vitamins
A series of Gram-negative standard strains, A. baumannii (ATCC 19606 and ATCC 17978), K. pneumoniae (ATCC 51503) and P. aeruginosa PAO1 and PAO14, in addition to some clinical strong biofilm producers of Acinetobacter (A5, A20 and A21), Klebsiella (K25, K27 and K28), and Pseudomonas (P8, P16, P24 and P27), were selected for testing the antimicrobial activity of vitamins D and K1.For vitamin D, the MIC was 1250 µg/ml for all the isolates, except for A. baumannii ATCC 17978 and K. pneumoniae ATCC 51503 standard strains was 625 µg/ ml.On the other hand, vitamin K1 had an MIC value of 2500 µg/ml for the majority of the tested isolates, and 5000 µg/ml was observed for only A. baumannii ATCC 17978, A21, K. pneumoniae K25 and K28, as well as P. aeruginosa PAO1, P8 and P24.Concentrations of vitamins below their MICs (1/2 and 1/4 MICs) were also calculated as described in Table 2.

Effect of vitamins on bacterial growth and viability
The effect of vitamins D and K1, at sub-MICs, on bacterial growth kinetics was studied against three isolates per genus, A5, K28 and P16.As indicated in Fig. ( 4a,  b, c), A5, K28 and P16 subjected to 1/2 MIC vitamin D and vitamin K1 grew at a slightly lower rate than did the vitamin-unexposed isolates.However, no significant difference in the final OD600 nm was noted between cultures with untreated broth and their counterparts with broth containing 1/2 MIC of the tested vitamins, indicating that the presence of vitamin D or vitamin K1 at sub-MICs did not hamper the bacterial growth over 24 h.
To complement the previous growth curve data, viable bacteria were also counted.Again, no considerable reduction in log CFU/mL was observed for either pathogen after 1/2 MIC exposure to vitamin D or vitamin K1 (Fig. 4d, e, f).

Effects of vitamin D and vitamin K1 on bacterial attachment
The CV assay was employed to assess the antiadherence and subsequent biofilm formation capacity of vitamin D and vitamin K1 at their respective sub-MICs against the tested standard and the previously selected strong biofilm-producing isolates.Compared to the dense biofilm formed by untreated cultures, vitamin D significantly reduced biofilm formation by 44-85% at 1/2 MIC and by 30-87% at 1/4 MIC in A. baumannii (Fig. 5a).In K. pneumoniae, vitamin D revealed a significant decrease at 1/2 and 1/4 MIC by 53-77% and 57-82%, respectively (Fig. 5b).Similarly, all tested isolates of P. aeruginosa treated with sub-MICs of vitamin D significantly decreased the biofilm architecture in the range of 25-91%, except for only two isolates; P8 and P24 (at 1/4 MICs) showed poor biofilm eradication (Fig. 5c, P < 0.0001).

Molecular identification of biofilm-regulatory and housekeeping genes Polymerase chain reaction (PCR)
PCR detection of the genes responsible for biofilm formation, cusD, bssS and pelA, revealed that these genes were carried by the representative isolates A5, K28 and P16, respectively (Additional file 1. Supplementary Fig. 2a).Furthermore, the presence of the standard genes rpoB in A. baumannii (A5), ropD in K. pneumoniae (K28) and ropD in P. aeruginosa (P16) was confirmed via PCR.The results showed that each genus completely harboured its reference-specialized gene (Additional file 1. Supplementary Fig. 2b).

Gene expression by qPCR
The relative expression of biofilm-forming genes in cultures treated with 1/2 MIC of vitamin D or vitamin K1 compared to that in untreated controls was displayed in Fig. 9.The Ct values of each tested gene (cusD, bssS and pelA) were determined, and their relative amounts were then normalized to those of the reference genes rpoB, ropD and ropD in the same sample.In A. baumannii A5, both vitamin D and vitamin K1 significantly reduced cusD gene expression by 76% and 98%, respectively (Fig. 9a).Similarly, upon treatment of K. pneumoniae K28 with vitamins D or K1, the expression of bssS was significantly reduced by 100% and 99.7%, respectively (Fig. 9b).In addition, exposure of P. aeruginosa P16 to vitamins D and K1 caused significant downregulation of pelA, by 99% and 98%, respectively (Fig. 9c).

Discussion
Antibiotic resistance is a concerning and formidable phenomenon because, in addition to its contribution to increasing the rate of morbidity and mortality, it is also associated with biofilm formation in numerous pathogens, especially Gram-negative organisms [41].This study focused on three of the most common resistant strains encountered in hospitals, namely, A. baumannii, K. pneumoniae and P. aeruginosa.A total of 117 Gramnegative isolates were collected from different clinical sources; P. aeruginosa was the most prevalent pathogen, accounting for 47.9%, followed by K. pneumoniae (34.2%).The lowest incidence was for A. baumannii (17.9%), which is similar to the results recorded by Sundaram and coauthors (21%) [42] and Rabina and colleagues (20%) [43].The maximum prevalence of K. pneumoniae was observed in blood cultures (42.5%), as previously detected [44][45][46].Most of the A. baumannii and P. aeruginosa strains were isolated from wound samples (28.6% and 46.4%, respectively), which is consistent with the finding of previous studies by Ruh et al. and Farajzadeh et al. [45,47].Notably, only two isolates of P. aeruginosa were purified from ear swabs, as P. aeruginosa is an opportunistic pathogen that can enter through a pierced ear, causing severe middle ear infections [48,49] (Fig. 1, Additional file 1. Supplementary Table 1).
The antibiotic resistance pattern demonstrated a concerning level of resistance across all the collected strains toward almost all the antimicrobial agents tested (Fig. 2, Additional file 1. Supplementary Table 1).The resistance to the tested β-lactam antibiotics was excessively high, ranging from more than 60% against IPM in P. aeruginosa to complete resistance (100%) towards AMC in A. baumannii and K. pneumoniae.This could be due to the usual and widespread usage of β-lactams without proper consideration of disease severity or following microbiological diagnostic procedures [50].Similar findings have been reported in many previous studies [51][52][53][54].Additionally, high resistance to aminoglycosides and CIP was observed against all the tested isolates, which is in accordance with the findings of other studies [51,[55][56][57].
Additionally, most of the isolates were MDR (70.1%), with incidences of 81, 75 and 62.5% for A. baumannii, K. pneumoniae and P. aeruginosa, respectively (Fig. 2d).A similar MDR prevalence in A. baumannii and K. pneumoniae has been previously reported [58].The ability of microbes to utilize various resistance mechanisms to evade the effects of antibiotics and to overcome the emergence of high resistance to critical antimicrobial classes is a great challenge, as described by the WHO.This disaster also worsened because of prominent nonsusceptibility to carbapenem (imipenem, Fig. 2), which is considered the last resort antimicrobial agent reserved for fighting serious bacterial infections [59].Therefore, the increase in MDR phenotypes and treatment of patients infected with these bacteria pose significant challenges [60].Notably, the MDR incidence was significantly greater for A. baumannii isolated from urine specimens (29.4%) than for those isolated from other sources, which indicated that the treatment of urinary tract infections caused by this pathogen is a concerning issue.Furthermore, MDR P. aeruginosa was detected in 58% of the wound samples, suggesting that there was too much post-surgical antibiotic usage and hospital readmission.
Biofilm formation is an intrinsic weapon for pathogenic and opportunistic isolates in various clinical settings.In this study, nearly all the isolates (93.2%, Fig. 3) were moderate or strong biofilm producers, and this proportion was much greater than that reported in other studies [61] (62.73%) [62], (71.8%) and [63] (87.5%).Consequently, it is crucial to diagnose and treat infections as early as possible prior to biofilm development, as this approach could enhance the response to antimicrobial therapy [64,65].Even poor biofilm-producing phenotypes may still be risky during polymicrobial infections where they can be integrated into preexisting biofilms or provide a synergistic effect with other robust biofilm formers [66].
The majority of strong biofilm producers were extracted from wounds (32.2%) more than from blood (25.4%) or other clinical sources, supporting the fact that that surgical wounds, skin lesions or any abrasion in mammalian tissues due to implanted medical devices favours the formation and evolution of bacterial biofilms [67,68].This result was consistent with those reported by Leshem et al. and Piechota et al. [69,70].MDR isolates had a more robust ability to form biofilms than did their counterparts, similar to the finding of Dumaru and colleagues [68].Biofilm architecture plays a crucial role in fortifying bacteria within a biofilm matrix against classical and even broad-spectrum antibiotics, allowing them to cause notorious and devastating diseases in healthy and immunocompromised individuals.Also, many researchers have shown a link between biofilm creation ability and resistance to certain antimicrobial agents [71][72][73].The emergence of small colony variants (SCVs), which remain viable inside the bacterial population at a slow growth rate, has been reported to be associated with special biofilm formation abilities and MDR [74].A bacterial switch to the SCV phenotype provides a significant colonization advantage, causing recurrent and aggressive infection.P. aeruginosa has been reported to produce SCV with an overproduction of EPS and a strong ability to form biofilms [75,76].Silva and coauthors reported the development of colistin-resistant biofilms in K. pneumoniae with SCV characteristics [77].
The capability of A. baumannii, K. pneumoniae and P. aeruginosa superbugs to develop biofilms provides insight into how these organisms escape antimicrobial therapies and cause life-threatening infections [11,78].Therefore, there is a need for effective approaches to tackle AMR.Hence, the interruption of Gram-negative biofilms by vitamin D and vitamin K1 at different concentrations was studied against some strong biofilm-producing isolates.Vitamins D and K1 possessed antimicrobial activity at concentrations ranging from 625 to 1250 µg/ ml and 2500-5000 µg/ml, respectively (Table 2).The antimicrobial effects of vitamin D3 on both Grampositive bacteria, such as S. aureus and S. pyogenes, and Gram-negative bacteria, including K. pneumoniae and E. coli were estimated [79].The reported antimicrobial activity of both vitamins D and K1 was associated with lipid solubility and alterations in membrane permeability [80].In the study of Andrade and colleagues, vitamin K3 was shown to possess antimicrobial activity, with an MIC of 64 µg/ml against P. aeruginosa 03, and to potentiate the antimicrobial activity of aminoglycosides [80].
The antimicrobial activity of both vitamins at the 1/2 MIC was monitored over a 24 h period by measuring the OD and CFU/ml of treated A. baumannii A5, K. pneumoniae K28 and P. aeruginosa P16 and comparing the obtained data with those of untreated cells cultivated under the same conditions (Fig. 4).Both the control and vitamin-challenged cultures grew exponentially, indicated by the lack of significant differences in the typical growth curves.Furthermore, vitamin D and vitamin K1 were not shown to have an effect on the CFU/ml of A5, K28 or P16 at 1/2 MIC.
The antibiofilm effects of vitamin D and vitamin K1 were phenotypically and genotypically assessed using concentrations of 1/2 MIC, or less (1/4 MIC), of both vitamins.Vitamins D and K1, below their MICs, significantly inhibited bacterial adhesion at 1/2 MIC more than 1/4 MIC, particularly among K. pneumoniae isolates (Figs. 5 and 6).Even after the biofilm of standard and clinical A. baumanni, K. pneumoniae and P. aeruginosa isolates was maturated after 24 h; vitamin D and K1 could effectively disrupt the biofilms and significantly reduce the bacterial biofilm density at concentrations of 0.5×, 1× and 2× the MIC (Figs. 7 and 8).This biofilm-hindering ability of both vitamins may be due to many factors that are responsible for forming and supporting the bacterial biofilm architecture, such as affecting EPS matrix production, bacterial motility, intra or intercellular communication or any other environmental conditions [81,82].Previous studies have evaluated different vitamin K types, including vitamins K1, K2 and K3.Soltani et al. elucidated the anti-quorum sensing and biofilm formation effects of vitamin K1, particularly through the inhibition of pqs pathway in P. aeruginosa [83].Supplementation with vitamin K3 decreased susceptibility to cell wall inhibitors and other antimicrobial agents, triggered EPS synthesis and subsequently increased the biofilm biomass content of S. aureus SCVs more than that of the wild-type S. aureus strain [84].Nonetheless, vitamin K3 has good antimicrobial and antibiofilm effects [85].
On the other hand, sub-MICs of some antimicrobial agents, such as aminoglycosides, increase biofilm formation by P. aeruginosa and E. coli [86,87].In addition, carbenicillin, tetracycline and colistin enhanced biofilm formation in E. coli and A. baumannii [88,89].Triclosan, a common bactericidal agent used in cosmetics, has short-term antibiofilm activity, as detected by Ricart and coauthors [90], and is typically tolerated by the bacterial community in the biofilm matrix within a few days [91].Carbapenems (imipenem, meropenem and doripenem) altered the biofilm morphology without affecting the viability of treated biofilms compared to untreated cells in K. pneumoniae [92] and induced the expression of alginate genes, leading to increased biofilm density in P. aeruginosa [93].Similarly, in Gram-positive bacteria, some β-lactams such as ampicillin, amoxicillin and cloxacillin, hinder the release of extracellular DNA which leads to increased biofilm volume in S. aureus [94] and E. faecalis [95].Besides, biofilm communities resist the destructive effect of conventional antibiotics because of the constant change in their EPS matrix [96].Therefore, antibiotics enhance biofilm evolution and resistance, leading to the environmental dissemination of resistant phenotypes and genotypes [97].
Previous studies have demonstrated the in vivo activity of vitamin D and its importance for normal intestinal homeostasis, with a reported effect on bacterial colonization [98].Moreover, vitamin D stimulates the production of antimicrobial peptides that maintain the barrier function of the gut microbiota and airway function [99].
Increasing the vitamin D concentration to more than 30-40 ng mL − 1 in human serum, reduces the risk of biofilm-associated caries [100].Additionally, vitamin D is a promising adjuvant therapy for managing biofilmrelated bacterial infections due to its immunomodulatory activity, as it can induce the secretion of cathelicidin, an antimicrobial peptide, while decreasing the secretion of inflammatory cytokines [101].Furthermore, vitamin K has a beneficial impact on the intestinal microbiota by preventing or treating intestinal disorders associated with microbial imbalances [102], including inflammatory bowel disorders [103].
To confirm the antibiofilm effect of both D and K1, the transcriptional response of the biofilm-regulatory genes cusD, bssS and pelA was assessed against the A5, K28 and P16 tested isolates, respectively.Compared with those in the corresponding nontreated cells, the expression levels of cusD in A5, bssS in K28 and pelA in P16 were markedly downregulated by 76-100% under both vitamin treatment conditions (Fig. 9).Specifically, vitamin D and vitamin K1 significantly reduced cusD gene expression in A. baumannii A5 by 76% and 98%, respectively (Fig. 9a).In A. baumannii, cusD is involved in cellular motility, surface adhesion and further biofilm formation [104].The overexpression of csuA/ABCDE is attributed to A. baumannii adherence, increased mushroom-like biofilm formation, and decreased bacterial motility [105].Therefore, the suppression of cusD gene could be associated with a reduction in bacterial colonization of surfaces and the subsequent inability to germinate biofilms.
Treatment of K. pneumoniae K28 with vitamins D and K1 significantly reduced the expression of bssS by 100% and 99.7%, respectively (Fig. 9b).Additionally, P. aeruginosa P16 treated with vitamins D and K1 exhibited significant downregulation of pelA expression by 99% and 98%, respectively (Fig. 9c).Both bssS and pelA are biofilm-associated small proteins that have a substantial functions in the synthesis and transport of EPS across K. pneumoniae and P. aeruginosa cells, respectively, thereby regulating biofilm maturation [106,107].Hence, suppression of pelA gene could be associated with the lack of pel polysaccharide-dependent biofilm formation, which is the primary exopolysaccharide biofilm matrix in P. aeruginosa [108].Similarly, the expression of biofilm-related genes in MRSA (icaA and icaR) and P. aeruginosa (lecA and pelA) was downregulated by vitamin K and vitamin C, respectively [15,109].
Therefore, this study provides prospective and promising potential for the usage of frequently consumed pharmaceutical supplements (vitamins D and K1) in the eradication of difficult-to-treat biofilm-triggered infections.However, potential antibiofilm endeavour of vitamins D and K1 were detected using representative A. baumannii, K. pneumoniae and P. aeruginosa isolates, and further research is needed to confirm these findings in other Gram-negative isolates and to identify other Gram-positive and MDR organisms.Additionally, in vivo analysis will provide deeper insights into the clinical applications of the obtained effects.

Conclusion
In summary, this work focused on studying the most common Gram-negative bacilli in hospital settings: A. baumannii, K. pneumoniae and P. aeruginosa.The significant increase in resistance, including MDR, and the increase in the prevalence of biofilm-forming isolates reflect an emerging threat posed by these superbugs in our country.Our results revealed that the two vitamins D and K1 exerted antimicrobial effects on the tested isolates at their pharmaceutical dosage.Moreover, they hindered biofilm attachment and disrupted its mature structure in the tested strains.This inhibitory effect was confirmed by the significant decrease in the expression of the genus-specific biofilm-encoding genes cusD, bssS and pelA.This antibiofilm property of both vitamins holds significant value in clinical therapies against the tested isolates.Coadministration of antibacterial agents with these vitamins could reduce or reverse resistance.However, further research is required to elucidate the in vivo activity of these vitamins.

Table 2 Minimal inhibitory concentrations (MICs) and subinhibitory concentrations (1/2 and 1/4 MICs) of the tested vitamins
This experimental study was supported by open access funding sponsored by "The Science, Technology & Innovation Funding Authority, STDF" in cooperation with The Egyptian Knowledge Bank (EKB).The study did not receive any other specific grant from funding agencies in the public, commercial, or nonprofit sectors.Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).